Fig. 6

The cell-surface-resident Cdo was specifically decreased in Stx4-depleted C2C12 cells. a C2C12 cells were transfected with pSuper or shStx4 expression vectors, and cells at the indicated differentiation time points were subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were also analyzed as control. b C2C12/pSuper or C2C12/shStx4 cells were transfected with Cdo-GFP expression vectors and subjected to immunostaining with N-Cadherin antibody, followed by confocal microscopy. The boxed area is shown as an enlarged view. The white arrows mark the area where the localization of Cdo-GFP under the N-Cadherin-resident cell surface is located. Size bar = 10 μm. c C2C12/pSuper or C2C12/shCdo cells were transfected with pcDNA or Stx4 expression vectors and subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were analyzed as control. d Control or shCdo-transfected C2C12 cells at D1 were subjected to surface biotinylation followed by streptavidin-bead pulldown and immunoblotting with indicated antibodies. e Control or shCdo expression vector transfected C2C12 cells were immunoprecipitated with antibody to GLUT4 and immunoblotted with antibodies to GLUT4, Stx4, and Cdo. f Stable C2C12 cells transfected with control, Cdo, or shCdo expression vector were incubated with or without 10 μg/ml insulin for 1 h, followed by 2-NBDG incubation for a further 1 h. Glucose uptake was measured by the relative fluorescence intensity. The experiment was repeated for three independent assays with similar results. Significant difference from insulin-incubated cells, *p < 0.05, **p < 0.01